This project aims at isolating mutant aminoacyl tRNA synthetases with altered recognition. The gene for Ala-tRNA synthetase has been cloned into two pBR322 recombinant plasmids. A plasmid carrying alaS is mutagenized in vitro and then transformed into an appropriate recipient. Selection is done for cells with a phenotype that could be due to the presence of a misrecognition mutant of Ala-tRNA synthetase. A number of mutants have been obtained. And an Ala-tRNA synthetase which recognizes tRNAI1e has been isolated. Various mutants that have already been isolated are to be characterized thoroughly and more mutants are to be prepared. These efforts should yield a variety of mutant Ala-tRNA synthetases having different specific tRNA recognition changes. Two plasmids carrying alaS have been partially mapped with restriction enzymes, and this analysis will continue. The alaS segment of one of these plasmids will be sequenced; from this, and from a limited amount of peptide sequencing, the entire amino acid sequence of the protein will be determined. The positional location of mutations that cause tRNA specificity changes will be identified. This information will enable us to test and to build upon existing ideas on how recognition is achieved and on the structural organization of tRNA binding regions.